Methods of treating autoimmune, respiratory and inflammatory disorders by inhalation of roflumilast n-oxide

ABSTRACT

The present disclosure relates to pharmaceutical compositions useful for (and to a method of) treating autoimmune, respiratory and/or inflammatory diseases and conditions. The method involves administering to a subject in need thereof roflumilast N-oxide by inhalation. The present disclosure particularly relates to the treatment of asthma and chronic obstructive pulmonary disease (COPD) by administering roflumilast N-oxide by inhalation.

The present application claims the benefit of Indian Patent ApplicationNos. 354/CHE/2013, filed Jan. 28, 2013, and 355/CHE/2013, filed Jan. 28,2013, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of treating an autoimmune,respiratory and/or inflammatory disease or condition, such as asthma,COPD and other allergic and/or inflammatory disorders of the lung bypulmonary administration (e.g., by inhalation) of roflumilast N-oxide ora pharmaceutically acceptable salt thereof. The invention also relatesto pharmaceutical compositions for use in the method.

BACKGROUND OF THE INVENTION

Autoimmune, respiratory and inflammatory diseases such as chronicobstructive pulmonary disorder (COPD) and asthma are chronic and oftenprogressive diseases associated with a dysregulated or overactive immunesystem.

Asthma is the most common chronic disease among children and alsoaffects millions of adults. Some 235 million people worldwide sufferfrom this disease.

COPD is a highly prevalent condition and a major cause of morbidity andmortality worldwide. As the disease progresses, patients with COPD maybecome prone to frequent exacerbations resulting in patient anxiety,worsening health status, lung function decline and increase in mortalityrate. These episodes of worsening respiratory function lead to increasesin health care utilization, hospital admissions and costs. Worse,frequent exacerbations are associated with a faster decline in lungfunction, thereby shortening life expectancy.

In addition to COPD and Asthma, other allergic and/or inflammatorydisorders of lung include diseases such as Cystic Fibrosis andIdiopathic pulmonary fibrosis (IPF).

According to the recommendations of Global Initiative for ChronicObstructive Lung Disease (GOLD), the first line therapy for COPD arelong acting β-agonists, long acting muscarinic antagonist and inhalationcorticosteroids. However, these drugs reduce the symptoms andexacerbations associated with the disease rather than targeting itsmolecular and cellular basis. Accordingly, there is still a need forfurther improvement in COPD therapy.

Roflumilast (Daliresp®), a PDE4 inhibitor, is approved as an oraltherapy in the U.S. to reduce the risk of COPD exacerbations in patientswith severe COPD associated with chronic bronchitis and a history ofexacerbations.

In April 2010, the Pulmonary-Allergy Drugs Advisory Committee (PADAC) tothe FDA voted 10 to 5 against approving roflumilast due to modestbenefit and potential for adverse events. In March 2011, the FDAapproved roflumilast with a narrower indication than had originally beenpursued (namely, the treatment to reduce the risk of COPD exacerbationsin patients with severe COPD associated with chronic bronchitis and ahistory of exacerbations). Roflumilast has been reported to have dosedependent toxicity, which limits the use of roflumilast at higher doses.The table below shows adverse events (AEs) that occurred in at least 2%of those receiving roflumilast and greater than placebo.

Roflumilast Placebo AEs (n = 4438) (n = 4192) Diarrhoea 420 (9.5) 113(2.7)  Weight loss 331 (7.5) 89 (2.1) Nausea 209 (4.7) 60 (1.4) Headache195 (4.4) 87 (2.1) Back pain 142 (3.2) 92 (2.2) Influenza 124 (2.8) 112(2.7)  Insomnia 105 (2.4) 41 (1.0) Dizziness  92 (2.1) 45 (1.1)Decreased  91 (2.1) 15 (0.4) appetite

-   -   Data obtained from the FDA label for Dalresp® (roflumilast) Aug.        13, 2013

R. W. Chapman et al., European Journal of Pharmacology, 571, 215-221(2007), report experiments involving administration of roflumilast byinhalation to Brown Norway rats in an attempt to improve its therapeuticindex.

According to the label for Daliresp, roflumilast N-oxide is an activemetabolite of roflumilast. International Publication Nos. WO 2001/90076and WO 2011/163469, both of which are hereby incorporated by reference,disclose the preparation and certain uses of roflumilast N-oxide.Additional efficacy, preclinical and clinical information forroflumilast and roflumilast N-oxide is provided in A. Hatzelmann et al.,Journal of Pharmacology and Experimental Therapeutics, 297, 267-279,2001; Center For Drug Evaluation And Research Pharmacology Review(s) onRoflumilast (Application Number: 022522Orig1s000) available online onthe U.S. FDA website; D. S. Bundschuh et al., Journal of Pharmacologyand Experimental Therapeutics, 297, 280-290, 2001; Rabe et al., Br. J.Pharmacol., 16353-67, 2011; Zuzana Diamant et al., PulmonaryPharmacology & Therapeutics 24, 4 (2011) 353; and S. Vollert et al.,Diabetologia, 55, 2779-2788, 2012, each of which is hereby incorporatedby reference.

Despite currently available intervention therapies, respiratorydisorders such as asthma and COPD remain a disease class with asignificant unmet medical need. More effective therapies with feweradverse events are needed.

SUMMARY OF THE INVENTION

The present invention provides a method of treating an autoimmune,respiratory and/or inflammatory disease and/or condition by pulmonaryadministration (e.g., by inhalation), an effective amount of roflumilastN-oxide or a pharmaceutically acceptable salt thereof. Without beingbound by any particular theory, the inventors theorize that pulmonaryadministration of roflumilast N-oxide will result in lower plasma levelsof roflumilast N-oxide and roflumilast and therefore fewer side effectsthan oral delivery of roflumilast. Furthermore, pulmonary delivery ofroflumilast N-oxide has a broader therapeutic window than oral deliveryof roflumilast. This permits a lower dosage of drug to be administeredand/or a longer drug regimen with fewer adverse events.

The disease or condition can be asthma, COPD, chronic obstructivebronchiolitis, chronic bronchitis, or allergic or non-allergic rhinitis.In a preferred embodiment, the disease or condition is asthma or COPD.

The roflumilast N-oxide (or pharmaceutically acceptable salt thereof)can be administered by inhalation in the form of a dry powder, solutionor suspension. In one embodiment, the roflumilast N-oxide isadministered as a dry powder. In another embodiment, the roflumilastN-oxide is administered as a solution or suspension. The roflumilastN-oxide may be administered, for example, using a metered dose inhaler(MDI) or a dry powder inhaler (DPI). Alternatively, the roflumilastN-oxide may be administered with a nebulizer (e.g., an ultrasonicnebulizer).

In one embodiment, the roflumilast N-oxide is administered as a singledose of about 5 μg to about 2000 μg. In another embodiment, theroflumilast N-oxide is administered as a single dose of about 20 μg toabout 1200 μg. One or more doses of the roflumilast N-oxide may beadministered a day.

In yet another embodiment, the roflumilast N-oxide is administered as asingle dose of about 50 μg to about 1000 ug, such as a single dose ofabout 100 μg to about 800 μg, for example, as a single dose of about 100μg, about 200 μg, about 400 μg or about 600 μg.

The present invention also relates to a pharmaceutical compositionsuitable for pulmonary administration (e.g., by inhalation) comprisingroflumilast N-oxide and optionally one or more pharmaceuticallyacceptable carriers and/or excipients. The pharmaceutical compositionmay be used in the methods of treatment described herein, such as forthe treatment of asthma or COPD.

The pharmaceutical composition may be in the form of an inhalable drypowder comprising roflumilast N-oxide as an active ingredient andoptionally particles of a physiologically acceptable,pharmacologically-inert solid carrier. In one preferred embodiment, theroflumilast N-oxide particles are in micronized form.

Yet another embodiment is a pharmaceutical composition in the form of anaerosol suitable for pulmonary administration comprising roflumilastN-oxide or a pharmaceutically acceptable salt thereof as an activeingredient, a propellant, and optionally one or more co-solvents,pharmaceutically acceptable carriers and/or excipients.

Yet another embodiment is a pharmaceutical composition suitable forpulmonary administration comprising a suspension of particles ofroflumilast N-oxide or a pharmaceutically acceptable salt thereof (e.g.,micronized particles of roflumilast N-oxide) in a propellant.

Yet another embodiment is a metered dose inhaler (MDI) or a dry powderinhaler (DPI) comprising a pharmaceutical composition comprisingroflumilast N-oxide.

In any of the methods or compositions described herein, the roflumilastN-oxide can have a particle size (for example, d₅₀ or d₉₀) of less thanabout 10 microns. In a preferred embodiment, the roflumilast N-oxide hasa mass median diameter, d₅₀, or d₉₀ equal to or less than about 10microns, preferably less than about 6 microns and more preferably fromabout 1 to about 6 microns. In yet another embodiment, the roflumilastN-oxide has a mass median particle size, d₅₀, or d₉₀ of less than about10 microns (e.g., from about 0.1 to about 10 microns, such as about 0.5to about 5 microns).

The roflumilast N-oxide may be in the form of an anhydrate, a solvate, ahydrate or a salt with a pharmacologically acceptable acid or base. In afurther embodiment of any of the methods or compositions describedherein, the roflumilast N-oxide may be in combination with a secondactive agent, such as one or more of leukotriene receptor antagonistsincluding LTD4-antagonists, corticosteroids, H1 receptor antagonists, β2adrenoceptor agonists, COX-2 selective inhibitors, statins,non-steroidal anti-inflammatory drugs (NSAIDs), M2 and/or M3antagonists, betamimetics, additional PDE4-inhibitors, EGFR-inhibitors,CCR3-inhibitors, iNOS-inhibitors, SYK-inhibitors, glucocorticoids, δ2agonists, p38 kinase inhibitors, NK1 receptor antagonists or anycombination of any of the foregoing.

In a preferred embodiment of any of the methods or compositionsdescribed herein, the roflumilast N-oxide is in combination with asecond active agent selected from long-acting β2 agonists (LABA), M3antagonists, corticosteroids, and any combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a scatter graph showing the effect of orally administeredvehicle and roflumilast (RFL) (0.3, 1, 3, and 10 mg/kg) on theinhibition of LPS-induced neutrophilia in the bronchoalveolar lavagefluid (BALF) of female Wistar rats (Example 3B.A).

FIG. 2 is a scatter graph showing the effect of intratracheally (IT)administered roflumilast (RFL) (10, 30, and 100 μg/kg) on the inhibitionof LPS-induced neutrophilia in the BALF of female Wistar rats (Example3B.A).

FIG. 3 is a scatter graph showing the effect of intratracheally (IT)administered roflumilast N-oxide (N-oxide) (10, 30, and 100 μg/kg) onthe inhibition of LPS-induced neutrophilia in the BALF of female Wistarrats (Example 3B.A).

FIGS. 4A and 4B are scatter graphs showing the effect of orallyadministered roflumilast (RFL) (1, 3, and 10 mg/kg b.i.d.) observed incigarette smoke induced cellular infiltration in BALB/c mice (Example3B.B).

FIGS. 5A and 5B are scatter graphs showing the number of macrophages andneutrophils, respectively, in a 4 day cigarette smoke induced cellularinfiltration COPD model following intranasally administered roflumilastN-oxide (N-oxide) (0.003, 0.03, 0.3, and 3 mg/kg intranasally) in BALB/cmice (Example 3B.B).

DETAILED DESCRIPTION OF THE INVENTION

The methods of present invention allow for the treatment of respiratoryand inflammatory diseases and conditions using a smaller amount ofactive compound and/or allow for the treatment of respiratory andinflammatory diseases and conditions for a longer period of time in amore efficient manner. The methods of present invention also allow forlower systemic side effects of roflumilast or roflumilast N-oxide thanwould have been expected upon oral administration of roflumilastN-oxide.

Roflumilast N-oxide has the formula:

The roflumilast N-oxide may be in the form of a pharmaceuticallyacceptable salt. Suitable pharmaceutically acceptable salts include, butare not limited to, salts derived from inorganic bases such as Li, Na,K, Ca, Mg, Fe, Cu, Zn, and Mn; salts of organic bases such asN,N′-diacetylethylenediamine, glucamine, triethylamine, choline,hydroxide, dicyclohexylamine, metformin, benzylamine, trialkylamine, andthiamine; salts of chiral bases such as alkylphenylamine, glycinol, andphenyl glycinol; salts of natural amino acids such as glycine, alanine,valine, leucine, isoleucine, norleucine, tyrosine, cystine, cysteine,methionine, proline, hydroxy proline, histidine, omithine, lysine,arginine, and serine; quaternary ammonium salts of the compounds ofinvention with alkyl halides, alkyl sulphates such as MeI and (Me)₂SO₄;salts of non-natural amino acids such as D-isomers or substituted aminoacids; salts of guanidine; and salts of substituted guanidine whereinthe substituent's are selected from nitro, amino, alkyl, alkenyl,alkynyl, ammonium or substituted ammonium salts and aluminum salts.Suitable salts may also include acid addition salts where appropriate,such as sulphates, nitrates, phosphates, perchlorates, borates,hydrohalides, acetates, tartrates, maleates, citrates, fumarates,succinates, palmoates, methane sulphonates, benzoates, salicylates,benzenesulfonates, ascorbates, glycerophosphates, and ketoglutarates.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary from, for example, between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) includes thoseembodiments, for example, an embodiment of any composition of matter,composition, method, or process, or the like, that “consist of” or“consist essentially of” the described features.

The term “effective amount” or “therapeutically effective amount” refersto that amount of roflumilast N-oxide described herein that issufficient to effect the intended application including, but not limitedto, disease treatment, as defined below. The therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated, e.g., theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells, e.g.,reduction of platelet adhesion and/or cell migration. The specific dosewill vary depending on the particular method of administration ofroflumilast N-oxide by inhalation chosen (e.g., by nebulization, aerosolfor inhalation or dry powder for inhalation), the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to which it isadministered, and the physical delivery system in which it is carried.

As used herein, the terms “treatment” and “treating” refer to anapproach for obtaining beneficial or desired results including, but notlimited to, therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

A “therapeutic effect,” as used herein encompasses a therapeutic benefitand/or a prophylactic benefit as described above. A prophylactic effectincludes delaying or eliminating the appearance of a disease orcondition, delaying or eliminating the onset of symptoms of a disease orcondition, slowing, halting, or reversing the progression of a diseaseor condition, or any combination thereof.

The term “subject” or “patient” as used herein refers to any animal,such as a mammal, for example a human. The methods and compositionsdescribed herein can be useful in both human therapeutics and veterinaryapplications. In some embodiments, the patient is a mammal, and in someembodiments, the patient is human. For veterinary purposes, the terms“subject” and “patient” include, but are not limited to, farm animalsincluding cows, sheep, pigs, horses, and goats; companion animals suchas dogs and cats; exotic and/or zoo animals; laboratory animalsincluding mice, rats, rabbits, guinea pigs, and hamsters; and poultrysuch as chickens, turkeys, ducks, and geese.

“Inflammatory response” as used herein is characterized by redness,heat, swelling and pain (i.e., inflammation) and typically involvestissue injury or destruction. An inflammatory response is usually alocalized, protective response elicited by injury or destruction oftissues, which serves to destroy, dilute or wall off (sequester) boththe injurious agent and the injured tissue. Inflammatory responses arenotably associated with the influx of leukocytes and/or leukocyte (e.g.,neutrophil) chemotaxis. Inflammatory responses may result from infectionwith pathogenic organisms and viruses, noninfectious means such astrauma or reperfusion following myocardial infarction or stroke, immuneresponses to foreign antigens, and autoimmune diseases. Inflammatoryresponses amenable to treatment with the methods and Roflumilast N-oxideaccording to the invention encompass conditions associated withreactions of the specific defense system as well as conditionsassociated with reactions of the non-specific defense system.

The methods of the present invention include methods for the treatmentof conditions associated with inflammatory cell activation.“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatibility antigens or cell adhesion molecules) ininflammatory cells (including, but not limited to, monocytes,macrophages, T lymphocytes, B lymphocytes, granulocytes(polymorphonuclear leukocytes including neutrophils, basophils, andeosinophils) mast cells, dendritic cells, Langerhans cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory condition.

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from an allergy.

“Arthritic disease” as used herein refers to any disease that ischaracterized by inflammatory lesions of the joints attributable to avariety of etiologies.

The pharmaceutical compositions of the invention may include acorticosteroid. Similarly, the methods described herein may includeco-treatment and/or co-administration with a corticosteroid. Therofumilast N-oxide and corticosteroid may be in the same composition orin separate compositions which are co-administered. Suitablecorticosteroids include, but are not limited to, dexamethasone,fluticasone, fluticasone furoate, prednisolone, betamethasone,budesonide, mometasone, mometasone furoate, triamcinolone acetonide,ciclesonide, TPI-1020, beclomethasone, beclomethasone dipropionate,prednisone, deflazacort, hydrocortisone, QAE-397, flunisolide, and anycombination thereof.

The pharmaceutical compositions of the invention may include a secondcomponent, such as one or more LABAs (long-acting β₂ agonists), M3antagonists, corticosteroids, and any combination of any of theforegoing. Similarly, the methods described herein may includeco-treatment and/or co-administration with such a second component. Therofumilast N-oxide and second component may be in the same compositionor in separate compositions which are co-administered.

The present invention also provides a method for the prevention ortreatment of any disease wherein the activity of PDE4 receptors isimplicated and inhibition of PDE4 receptor activity is desired, whichmethods comprise administering to a patient in need thereof atherapeutically effective amount of Roflumilast N-oxide alone, or incombination with a second compactive agent selected from LABA(long-acting β₂ agonists), M3 antagonists and/or corticosteroids.

The diseases wherein the activity of PDE4 receptors and inhibition ofPDE4 receptors are implicated include, e.g., diseases of the respiratorytract, characterized by airway obstruction, such as asthma and COPD.

The present invention is also directed to a device which may be asingle- or multi-dose dry powder inhaler, a metered dose inhaler or asoft mist nebulizer comprising Roflumilast N-oxide.

The present invention is also directed to a kit comprising apharmaceutical composition of Roflumilast N oxide, alone or incombination with an additional active ingredient, in admixture with oneor more pharmaceutically acceptable carriers and/or excipients, and adevice which may be a single- or multi-dose dry powder inhaler, ametered dose inhaler or a soft mist nebulizer.

In the airways, the physiological responses to elevated intracellularlevels of cyclic nucleotides, in particular of cAMP, lead to thesuppression of the activity of immune and pro-inflammatory cells such asmast cells, macrophages, T lymphocytes, eosinophils and neutrophils,resulting in a decrease of the release of inflammatory mediators whichinclude cytokines such as IL-I, IL-3 and tumor necrosis factor-alpha(TNF-α). It also leads to an airway smooth muscle relaxation and adecrease in oedema.

PDE-4 inhibitors, such as roflumilast N-oxide, exhibit an in vitroinhibitory activity toward the PDE4 enzyme in the nM range and exhibit aremarkable activity in the lungs upon intratracheal administration in ananimal model of COPD. They may also exhibit sustained pulmonary levelsin the lungs, being undetectable in plasma, which is an index of a shortsystemic action.

For the treatment of the diseases of the respiratory tract as providedherein, roflumilast N-oxide is administered by inhalation. Inhalablepreparations include, for example, inhalable powders,propellant-containing metering aerosols and propellant-free inhalableformulations.

For administration as a dry powder, a known single- or multi-doseinhalers may be utilized. The dry powder may be filled in gelatine,plastic or other capsules, cartridges or blister packs or in areservoir. A diluent or carrier, generally chemically inert toRoflumilast N-oxide, e.g., lactose or any other additive suitable forimproving the respirable fraction may be added to the powdered compoundsof the invention.

Inhalation aerosols containing propellant gas such as hydrofluoroalkanesmay contain the rofumilast N-oxide either in solution or in dispersedform. The propellant-driven formulations may also contain otheringredients such as co-solvents, stabilizers and optionally otherexcipients.

The propellant-free inhalable formulations comprising roflumilastN-oxide may be in form of solutions or suspensions in an aqueous,alcoholic or hydroalcoholic medium and they may be delivered by knownjet or ultrasonic nebulizers or by soft-mist nebulizers such asRespimat®.

Roflumilast N-oxide may be administered as the sole active agent or incombination with one or more other pharmaceutical active ingredientsincluding those currently used in the treatment of respiratorydisorders, such as for example, but not limited to, β₂-agonists,corticosteroids and M3 antagonists.

The dosages of roflumilast N-oxide may depend upon a variety of factorsincluding the particular disease to be treated, the severity of thesymptoms, the frequency of the dosage interval, rofulumilast N-oxideutilized, the efficacy, toxicology profile, and pharmacokinetic profileof rofulumilast N-oxide.

Advantageously, rofulmilast N-oxide may be administered by theinhalation route. The dosage of roflumilast N-oxide is preferablybetween about 0.01 and about 20 mg/day, such as between about 0.1 andabout 10 mg/day. More preferably, the dosage is between about 0.1 andabout 5 mg/day.

Preferably, rofulmilast N-oxide alone or combined with other activeingredients may be administered for the prevention, treatment,inhibition, or suppression of any obstructive respiratory disease suchas asthma, chronic bronchitis and chronic obstructive pulmonary disease(COPD).

Rofulmilast N-oxide may be administered for the prevention and/ortreatment of any disease wherein the activity of PDE4 enzyme isimplicated and inhibition of PDE4 enzyme activity is desired, or adisease state which is mediated by PDE4 activity (for instance a diseasestate in which PDE4 is overexpressed or overactive).

Examples of such diseases include, but are not limited, allergic diseasestates such as atopic dermatitis, urticaria, allergic rhinitis, allergicconjunctivitis, vernal conjunctivitis, eosinophilic granuloma,psoriasis, inflammatory arthritis, rheumatoid arthritis, septic shock,ulcerative colitis, Crohn's disease, reperfusion injury of themyocardium and brain, chronic glomerulonephritis, endotoxic shock,cystic fibrosis, idiopathic pulmonary fibrosis (IPF) arterialrestenosis, atherosclerosis, keratosis, rheumatoid spondylitis,osteoarthritis, pyresis, diabetes mellitus, pneumoconiosis, toxic andallergic contact eczema, atopic eczema, seborrheic eczema, lichensimplex, sunburn, itching in the anogenital area, alopecia areata,hypertrophic scars, discoid lupus erythematosus, systemic lupuserythematosus, follicular and wide-area pyodermias, endogenous andexogenous acne, acne rosacea, Beghet's disease, anaphylactoid purpuranephritis, inflammatory bowel disease, leukemia, multiple sclerosis,gastrointestinal diseases, and autoimmune diseases.

Examples of such diseases also include neurological and psychiatricdisorders such as Alzheimer's disease, multiple sclerosis,amylolaterosclerosis (ALS), multiple systems atrophy (MSA),schizophrenia, Parkinson's disease, Huntington's disease, Pick'sdisease, depression, stroke, and spinal cord injury.

Pharmaceutical Compositions

One aspect of the present invention provides a pharmaceuticalformulation comprising roflumilast N-oxide in the form of an inhalabledry powder wherein the formulation comprises micronized particles of aroflumilast N-oxide as an active ingredient, and particles of aphysiologically acceptable pharmacologically-inert solid carrier. Priorto the present invention, it was not known that roflumilast N-oxidecould be prepared as micronized particles suitable for inhalation.

According to another aspect, the present invention provides a dry powderinhaler comprising an inhalable dry powder of any embodiment describedherein.

A further aspect of the present invention refers to an inhalable drypowder of the present invention for use for the prevention and/ortreatment of a disease where the PDE4 enzyme is implicated andinhibition of PDE4 enzyme activity is desired, or a disease state whichis mediated by PDE4 activity, and in particular use for the preventionand/or treatment of an inflammatory or obstructive airways disease suchas asthma or chronic obstructive pulmonary disease (COPD).

Another aspect of the present invention refers to a method ofpreventing, treating, inhibiting, or suppressing an inflammatory orobstructive airways disease such as asthma or chronic obstructivepulmonary disease (COPD), the method comprising administration byinhalation of a therapeutically effective amount of an inhalable drypowder according to any embodiment described herein.

Another aspect of the present invention is directed to a packagecomprising an inhalable dry powder formulation according to anyembodiment described herein and a dry powder inhaler.

By “single therapeutically effective dose” it is meant the quantity ofactive ingredient administered at one time by inhalation upon actuationof the inhaler. The “single therapeutically effective dose” may bedelivered in one or more actuations, preferably one actuation (shot) ofthe inhaler. For “actuation” it is meant the release of activeingredient from the device by a single activation (e.g. mechanical or bybreath).

In general terms, the particle size of particles is quantified bymeasuring a characteristic equivalent sphere diameter, known as volumediameter, by laser diffraction. The particle size can also be quantifiedby measuring the mass diameter by means of a suitable known instrumentsuch as, for instance, a sieve analyzer.

The volume diameter (VD) is related to the mass diameter (MD) by thedensity of the particles (assuming a size independent density for theparticles).

The particle size may be expressed in terms of mass diameter (MD) andthe particle size distribution is expressed in terms of: i) the massmedian diameter (MMD) which corresponds to the diameter of 50 percent byweight or volume respectively, of the particles, and ii) the MD inmicron of 10% and 90% of the particles, respectively. The terms MMD andmean particle size are used interchangeably. In particle sizemeasurements, d₉₀, d₅₀ and d₁₀ respectively mean that 90%, 50% and 10%of the material is less than the micron size specified.

Laser diffraction measurement of particle size can use a dry method(wherein a suspension of the compound/salt in an airflow crosses thelaser beam) or a wet method (wherein a suspension of the compound/saltin a liquid dispersing medium, such as isooctane (e.g. if compound issoluble in isooctane) or 0.1% Tween 80 in water, crosses the laserbeam). With laser diffraction, particle size may be measured, forexample, with a Malvern Mastersizer or Sympatec apparatus. For example,particle size measurement and/or analysis by laser diffraction can useany or all of (preferably all of) the following: a Malvern Mastersizerlongbed version, a dispersing medium of 0.1% Tween 80 in water, a stirrate of ca. 1500 rpm, ca. 3 mins sonification prior to final dispersionand analysis, a 300 RF (Reverse Fourier) lens, and/or the Fraunhofercalculation with Malvern software. Unless specified otherwise, all d50and d90 measurements are measured by laser diffraction using a wetmethod.

The expression “respirable fraction” refers to an index of thepercentage of active particles which would reach the deep lungs in apatient. The respirable fraction, also termed fine particle fraction, isevaluated using a suitable in vitro apparatus such as Multistage CascadeImpactor or Multi Stage Liquid Impinger (MLSI) according to proceduresreported in common Pharmacopeias. It is calculated by the ratio betweenthe delivered dose and the fine particle mass (formerly fine particledose). A respirable fraction higher than 30% is an index of goodinhalatory performance.

The delivered dose is calculated from the cumulative deposition in theapparatus, while the fine particle mass is calculated from thedeposition on Stages 3 (S3) to filter (AF) corresponding to particles<4.7 microns. The expression “accurate therapeutically active dose ofthe active ingredient” refers to a formulation wherein the variationbetween the mean delivered daily dose and the mean emitted dose is equalto or less than 15%, preferably less than 10%.

In one aspect, the compositions of the invention are pharmaceuticalformulations in the form of inhalable dry powder comprising micronizedparticles of a Roflumilast N-oxide and particles of a physiologicallyacceptable pharmacologically-inert solid carrier.

The compositions according to the invention comprise the activeingredient in an amount such that, in case of administration byinhalation from inhalers, the therapeutically effective single dose(hereinafter the single dose) of a roflumilast N-oxide comprises betweenabout 5 μg and about 2000 μg, such as between about 20 μg and about 1200μg, e.g., between about 50 μg and about 1000 μg, between about 100 μgand about 800 μg or between about 100 μg, and about 600 μg.

According to a preferred embodiment, the single dose may be betweenabout 100 and about 300 μg, while according to another preferredembodiment; the single dose may be comprised between about 200 and about800 μg, more preferably between about 300 ug and about 600 μg. In otherembodiments, the single dose may be about 100 μg, about 200 μg, about400 μg or about 600 μg.

The single dose will depend on the kind and the severity of the diseaseand the conditions (weight, sex, age) of the patient and shall beadministered one or more times a day, preferably once or twice a day.

The daily dose at which the pharmaceutical composition comprising aRoflumilast N-oxide shall be comprised between about 100 μg and about2000 μg, preferably between about 200 μg and about 1000 μg, morepreferably between about 200 μg and about 800 μg and more preferablybetween about 100 μg and about 600 μg.

In one embodiment, the daily dose may be reached by a single or doubleadministration.

In another embodiment, the daily dose may be reached by a singleadministration and delivered in one actuation of the inhaler.

In another embodiment, the daily dose may be reached by a singleadministration and delivered in more than one actuation of the inhaler,preferably two actuations

In another embodiment, the daily dose may be reached by a doubleadministration and delivered in one actuation of the inhaler.

In another embodiment, the daily dose may be reached by a doubleadministration and delivered in more than one actuation of the inhaler,preferably two actuations.

The particles of Roflumilast N-oxide in the formulations according tothe present invention are preferably in a finely divided (micronized)form, i.e. their mass median diameter should generally be equal to orless than about 10 microns, preferably less than about 6 microns, morepreferably comprised between about 1 and about 6 microns.

The active ingredient may be produced in the desired particle size usingknown methods, e.g., by milling, direct precipitation, spray-drying,freeze-drying or supercritical fluids.

The carrier particles may be made of any physiologically acceptablepharmacologically-inert material or combination of materials suitablefor inhalatory use.

For example, the carrier particles may be selected from sugar alcohols;polyols, for example sorbitol, mannitol and xylitol, and crystallinesugars, including monosaccharides and disaccharides; inorganic saltssuch as sodium chloride and calcium carbonate; organic salts such assodium lactate; and other organic compounds such as urea,polysaccharides, for example starch and its derivatives;oligosaccharides, for example cyclodextrins and dextrins.

Advantageously the carrier particles are made of a crystalline sugar,for example, a monosaccharide such as glucose or arabinose, or adisaccharide such as maltose, saccharose, dextrose or lactose.

The formulations described herein may be prepared according to knownmethods. Generally the process comprises the steps of:

i) micronising together the active ingredient and the carrier; and ii)subjecting the resulting co-micronized mixture to agglomeration andspheronisation.

Alternatively, the process comprises the following steps:

i) micronising separately the active ingredient and the carrier; ii)mixing the micronized components; and iii) subjecting the resultingmixture to agglomeration and spheronisation.

When a formulation of the invention is in form of an ordered mixture, itmay advantageously comprise an additive material able to promote therelease of the active particles from the carrier particles on actuationof the inhaler device, thereby improving the respirable fraction.

The additive material, which is preferably bound to the surface of thecarrier coarse particles, is of a different material from the carrierparticles.

The additive material may be an amino acid, preferably selected fromleucine, isoleucine, lysine, valine, methionine, and phenylalanine. Theadditive may be a salt of a derivative of an amino acid, for exampleaspartame or acesulfame potassium.

Alternatively, the additive material may include or be one or more watersoluble surface active materials, for example lecithin, in particularsoya lecithin.

In a particular embodiment of the invention, the additive material mayinclude or consist of one or more lubricant selected from the groupconsisting of stearic acid and salts thereof such as magnesium stearate,sodium lauryl sulphate, sodium stearyl fumarate, stearyl alcohol,sucrose monopalmitate.

Other possible additive materials include talc, titanium dioxide,aluminium dioxide, and silicon dioxide.

Advantageously, the additive particles have a starting mean particlesize or d₅₀ of less than about 35 microns. Preferably they have a meanparticle size or d₅₀ of not more than 1 about 5 microns, more preferablynot more than about 10 microns

The optimum amount of additive material depends on the chemicalcomposition and other properties of the additive material. In general,the amount of additive is not more than about 10% by weight, based onthe total weight of the formulation. In certain embodiments, the amountof additive material is not more than about 5%, preferably not more thanabout 2%, not more than about 1% by weight or not more than about 0.5%based on the total weight of the formulation. In general, the amount ofadditive material is at least 0.01% by weight based on the total weightof the formulation.

The formulations of the invention in the form of ordered mixture mayalso comprise fine particles of a physiologically acceptablepharmacologically-inert material with a mass median diameter (MMD) equalto or less than about 15 micron, preferably equal to or less than about10 microns, even more preferably equal to or less than about 6 microns.

The percentage of fine particles of physiologically acceptablepharmacologically-inert material is advantageously comprised betweenabout 0.1 and about 40% of the total amount of the formulation.

Preferably, the coarse particles and the fine particles are constitutedof the same physiologically acceptable pharmacologically-inert material.

A formulation in the form of an ordered mixture according to theinvention may be prepared according to known methods. Suitable methodsmay comprise the step of mixing together the carrier coarse particles,the optional fine carrier particles and the additive particles, andfinally adding the finely divided Roflumilast N-Oxide to the resultingmixture. A preferred formulation according to the invention may beprepared according to the methods reported in International PublicationNo. WO 2001/78693.

The presence of the additive material embedded in the microparticles maybe detected according to known methods, for instance, by electronscanning microscope coupled to microcalorimetry.

The formulations of the invention may further comprise other therapeuticagents useful for the prevention and/or treatment of a respiratorydisease, e.g. β2-agonists such as salbutamol, salmeterol, andvilanterol, corticosteroids such as fluticasone propionate or furoate,flunisolide, mometasone furoate, rofleponide and ciclesonide,anticholinergic or antimuscarinic agents such as ipratropium bromide,oxytropium bromide, tiotropium bromide, oxybutynin, and combinationsthereof.

The dry powder formulations described herein may be used in allcustomary dry powder inhalers, such as unit dose or multidose inhalers.

For example, the formulation of the invention may be filled in hardgelatine capsules, in turn loaded in a unit dose inhaler such as theAerolizer™. Alternative, the formulation as a powder may be filled in amultidose inhaler comprising a powder reservoir such as that describedin International Publication No. WO 2004/012801.

The invention also relates to any one of the formulations describedbefore, for use as a medicament.

In one aspect, the present invention provides a pharmaceuticalformulation suitable for aerosol administration by a Pressurized MeteredDose Inhaler (pMDI), (hereinafter refered to as a pMDI formulation,comprising roflumilast N-oxide and a propellant.

In a particular embodiment, the pMDI formulation may be in form ofsuspension of particles of a micronized roflumilast N-oxide in apropellant, so as to permit inhalation of the active ingredient into thelungs upon administration of the aerosol formulation.

Advantageously the particles of the active ingredient shall have a massmedian diameter (MMD) of less than about 10 microns, preferably in therange of about 1 to about 10 microns, more preferably between about 1and about 6 microns.

Any pressure-liquefied propellant may be used, preferably ahydrofluoroalkane (HFA) propellant. Suitable examples of HFA propellantsinclude, but are not limited to, 1,1,1,2-tetrafluoroethane (HFA 134a),1,1,1,2,3,3,3-heptafluoro-propane (HFA227) and mixtures thereof.

In certain embodiments the propellant may include HFA 134a, while inother embodiments, the propellant may include HFA 227, or a mixturethereof in any ratio.

In a particular embodiment the suspension pMDI formulations comprise asurfactant, which may also act as a valve lubricant.

Suitable surfactants are known in the art and include, for example,sorbitan esters such as sorbitan trioleate, sorbitan monolaurate,sorbitan mono-oleate and their ethoxylated derivates such as polysorbate20, polysorbate 80; ethylene oxide/propylene oxide co-polymers and otheragents such as natural or synthetic lecithin, oleic acid,polyvinylpyrrolidone (PVP), preferably PVP (K25) and polyvinyl alcohol,olive oil, glyceryl monolaurate, corn oil, cotton seed oil or sunflowerseed oil, isopropyl myristate, oleyl alcohol, polyoxyethylene (20)sorbitan monolaurate, polyoxy ethylene (20) sorbitan mono-oleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, block co-polymers of oxyethylene andoxypropylene, diethylene glycol dioleate, tetrahydro fur fury 1 oleate,ethyl oleate, glyceryl mono-oleate, glyceryl monostearate, glycerylmonoricinoleate, cetyl alcohol, stearyl alcohol, cetyl pyridiniumchloride, ethylene oxide/propylene oxide co-polymer and ethoxylatedalcohols such as polyethylene glycol (PEG) 300-1000, diethylene glycolmonoethyl ether, Antarox, Brij, and any combination of the foregoing.

The amount of surfactant, which may be present in the pMDI formulationaccording to the invention, is usually in the range of about 0.001 toabout 3.0% (w/w), preferably between about 0.005 and about 1.0% (w/w).

Optionally, the pMDI formulation may contain a co-solvent. Suitableco-solvents include, but are not limited to, polar compounds thatcontain one or more hydroxyl groups or other polar groups. For example,suitable co-solvents include an alcohol, such as ethanol, preferablyanhydrous ethanol, isopropanol; a glycol such as propylene glycol,polyethylene glycol, polypropylene glycol or glycerol; a glycol ether;and a polyoxy ethylene alcohol, or any combination thereof.

In one embodiment, the co-solvent is anhydrous ethanol. In a preferredembodiment, the anhydrous ethanol is used in a concentration lower thanabout 20% (w/w), preferably below about 15%, more preferably betweenabout 1% and about 5% (w/w), most preferably about 1% (w/w) or about 5%(w/w).

In other embodiments, the pMDI formulations according to the inventionmay additionally include additional excipients. Examples of additionalexcipients include sugars such as lactose, amino acids such as alanine,betaine, cysteine, and/or antioxidants such as ascorbic acid, citricacid, sodium edetate, editic acid, tocopherols, butylhydroxytoluene,butylhydroxyanisol and ascorbyl palmitate.

The weight ratio of the drug to the excipient is generally in the rangefrom about 1:0.1 to about 1:100.

The pharmaceutical pMDI formulation of the invention may containRoflumilast N-oxide in an amount between about 0.02 and about 0.7% w/w,preferably between about 0.05 and about 0.5%, a co-solvent in an amountbetween about 1 and about 5% w/w, and one or more surfactants in anamount between about 0.001% and about 3% w/w.

To prepare the suspension pMDI formulation according to the invention,Roflumilast N-oxide is micronized by methods known in the art, toprepare the active substance in the form of particles having a typicalparticle size suitable for inhalation, such as a d₅₀ or d₉₀ less thanequal to 5 μm and more preferably such as less than equal to 3 μm.

According to another aspect, the present invention provides a pMDIcomprising a canister filled with a pharmaceutical formulation of thepresent invention and a metering valve for delivering a dailytherapeutically effective dose of the active ingredient.

The pMDI formulation of the invention is filled into pMDIs. The pMDIscomprise a canister fitted with a metering valve. Actuation of themetering valve allows a small portion of the spray product to bereleased to a subject.

In one embodiment, the formulation is actuated by a metering valvecapable of delivering a volume of between about 25 μl and about 100 μl,

Advantageously, the MDI device filled with the formulation may beequipped with a dose counter.

Conventional bulk manufacturing methods and known machinery may beemployed for the preparation of large scale batches for the commercialproduction of filled canisters.

For example, the pMDI suspension formulations according to the inventionmay be prepared by adding the active ingredient to a chilled propellantor optionally a pre-mixed blend of propellant and optionally furtherexcipients and, then dispersing the resulting suspension using asuitable mixer. After homogenization the suspension can be filled intothe MDI canister which is closed by crimping a metering valve on thecanister.

Alternatively, the active ingredient and optionally further excipientscan be added to a vessel. The liquefied propellant is then introducedinto the vessel under pressure and the active ingredient is dispersedand homogenized using a suitable mixer and homogenizer. Afterhomogenization the bulk formulation can be transferred into theindividual MDI canisters by using valve to valve transfer methods.

Alternatively, the co-solvent, if present, is introduced into a vesselat room pressure. The active ingredient and optional further excipientsare added and homogenised using a suitable homogenizer. The ethanolicsuspension is kept under stifling. The ethanolic bulk is then dosed intothe open canister. The valve is placed onto the can and crimped.Finally, the canister is pressure-filled with the final solutionformulation through the valve.

The pMDI formulations according to the invention, depending on volume ofthe metering valve to be used, may comprise from about 0.1 mg to about50 mg of Roflumilast N-oxide per ml, preferably from about 0.5 mg toabout 25 mg of Roflumilast N-oxide per ml.

The pMDI formulations in the form of suspensions comprising particles ofa micronized Roflumilast N-oxide and a propellant, comprise the activeingredient in an amount such that, in the case of administration byinhalation from inhalers, the daily therapeutically effective dose(hereinafter the daily dose) of Roflumilast N-oxide is between about 5μg and about 2000 μg, preferably between about 20 μg and about 1500 μg,even more preferably between about 50 μg and about 1000 μg, even morepreferably between about 60 μg and about 800 μg, even more preferablybetween about 200 μg and about 600 μg.

According to a preferred embodiment, the single dose is between about100 μg and about 300 μg. According to another preferred embodiment, thesingle dose is between about 200 μg and about 800 μg, more preferablybetween about 300 μg and about 600 μg.

In further embodiments, the single dose may be about 100 μg, about 200μg, about 400 μg or about 600 μg.

The single dose will depend on the kind and the severity of the diseaseand the conditions (weight, sex, age) of the patient and will beadministered one or more times a day, preferably once a day.

The daily dose may be delivered in one or two or more actuations (shots)of the inhaler wherein the pharmaceutical composition is contained. Forexample, a 400 μg daily dose may be administered in one shot of 400 μgor as two shots of 200 μg dose.

In another aspect, Roflumilast N-oxide may be dissolved or suspended togive a nebulised aqueous solution or suspension (herein called anebulised formulation), available either as for a single dose ormulti-dose vials formulation.

The nebulised formulation may have the pH and/or tonicity adjusted withsuitable buffers and/or isotonic agents, and optionally, it may alsocomprise stabilizing and/or preserving agents.

The present invention also provides a single dose or multidose vialfilled with a nebulised formulation as described herein for delivering adaily therapeutically dose of the active ingredient by a nebulizer.

A liquid, propellant-free pharmaceutical formulation in the form of aready-to-use preparation for administration by nebulisation of theinvention, comprises Roflumilast N-oxide in an amount such that thedaily dose is between about 35 μg and about 7000 μg, preferably betweenabout 70 μg and about 3500 μg, even more preferably between about 175 μgand about 2800 μg, even more preferably between about 280 μg and about2100 μg, even more preferably between about 350 μg and about 1750 μg.

According to a preferred embodiment, the single dose may be comprisedbetween about 350 μg and about 700 μg, while according to anotherpreferred embodiment, the single dose may be comprised between about 700μg and about 1400 μg.

In further embodiments, the single dose may be about 350 μg, about 700μg or 1400 μg.

The formulation is preferably used as a ready-to-use formulation.

In another embodiment, the nebulised formulation may also be in alyophilised form in unitary doses for the reconstitution in a solution.In this embodiment, a single dose of a lyophilised preparation may bereconstituted before use.

These nebulised formulations may also be distributed in suitablecontainers such as multidose vials or, preferably, single dose vials forsingle dosage administration. Said single-dose vials may bepre-sterilised or, preferably, may be aseptically filled using “blow,fill and seal” technology (see http://www.brevettiangela.com). Thefilling is preferably carried out under inert atmosphere.

Solution formulations can be advantageously sterilized by filtration.

The single-dose vials are preferably 2 ml volume. For suspensionformulations, the sterilization process is carried out through knowntechniques.

These formulations are intended for administration using suitablenebulizing apparatus such as jet nebulizers, ultrasonic nebulizers,mesh-vibrating nebulizers, soft-mist nebulizers such as Respimat® orothers.

The invention is also directed to a kit comprising a nebulisedformulation as described herein filled in vials for single dosageadministration and a nebulizer.

All the pMDI and nebulized formulations of the present invention mayfurther comprise other therapeutic agents, such as those used in thetreatment of respiratory disorders, e.g. corticosteroids such astriamcinolone acetonide, fluticasone propionate, fluticasone furoate,flunisolide, mometasone furoate, rofleponide and ciclesonide;anticholinergic or antimuscarinic agents such as ipratropium bromide,oxytropium bromide, glycopyrronium bromide and tiotropium bromide;long-acting β₂ agonists such as vilanterol, indacaterol, milveterol,salbutamol, levalbuterol, terbutaline, AZD-3199, BI-1744-CL, LAS-100977,bambuterol, isoproterenol, procaterol, clenbuterol, reproterol,fenoterol and ASF-1020 and salts thereof.

The invention also relates to any one of the formulations describedbefore, for use as a medicament.

In a further aspect, the present invention comprises any one of theformulations described before, for use in the prevention and/ortreatment of an inflammatory or obstructive airways disease such asasthma or chronic obstructive pulmonary disease (COPD).

In a further aspect, the present invention comprises the use of any oneof the formulations described before, in the prevention and/or treatmentof an inflammatory or obstructive airways disease such as asthma orchronic obstructive pulmonary disease (COPD).

In a still further aspect, the present invention comprises a method ofpreventing and/or treating an inflammatory or obstructive airwaysdisease such as asthma or chronic obstructive pulmonary disease (COPD),which comprises administration by inhalation of an effective amount ofone of the formulations described before.

Administration of all the formulations of the invention may be indicatedfor the prevention and/or treatment of mild, moderate or severe acute orchronic symptoms or for prophylactic treatment of respiratory diseasessuch as asthma and chronic obstructive pulmonary disease (COPD). Otherrespiratory disorders characterized by obstruction of the peripheralairways as a result of inflammation and presence of mucus such aschronic obstructive bronchiolitis and chronic bronchitis may alsobenefit by this kind of formulation.

A further embodiment of the present invention relates to apharmaceutical composition according to the present invention for use inthe treatment of respiratory and inflammatory diseases and conditions,particularly wherein the respiratory and inflammatory diseases orconditions are selected from asthma, allergic and non-allergic rhinitisand COPD.

In a further embodiment of any of the methods or compositions describedherein, the Roflumilast N-oxide is in combination with a second activeagent, selected from, for example, leukotriene receptor antagonistsincluding LTD4-antagonists, corticosteroids, H1 receptor antagonists, β2adrenoceptor agonists, COX-2 selective inhibitors, statins,non-steroidal anti-inflammatory drugs (“NSAIDs”), M2 and/or M3antagonists, betamimetics, additional PDE4-inhibitors, EGFR-inhibitors,CCR3-inhibitors, iNOS-inhibitors, SYK-inhibitors, glucocorticoids, δ2agonists, p38 kinase inhibitors, NK1 receptor antagonists and anycombination thereof.

Suitable β2-agonists for use in the present invention include, but arenot limited to, arformoterol, bambuterol, bitolterol, broxaterol,carbuterol, clenbuterol, dopexamine, fenoterol, formoterol,hexoprenaline, ibuterol, Isoetharine, isoprenaline, levosalbutamol,mabuterol, meluadrine, metaprotenerol, nolomirole, orciprenaline,pirbuterol, procaterol, reproterol, ritodrine, rimoterol, salbutamol,salmefamol, salmeterol, sibenadet, sotenerot, sulfonterol, terbutaline,tiaramide, tulobuterol, GSK-597901, GSK-159797, GSK-678007, GSK-642444,GSK-159802, HOKU-81, (−)-2-[7(S)-[2(R)-Hydroxy-2-(4-hydroxyphenyl)ethylamino]-5,6,7,8-tetrahydro-2-naphthyloxy]-N,N-dimethylacetamidehydrochloride monohydrate, carmoterol, QAB-149 and5-[2-(5,6-diethylindan-2-ylamino)-1-hydroxyethyl]-8-hydroxy-1H-quinolin-2-one,4-hydroxy-7-[2-{[2-{[3-(2-phenylethoxy)propyl]sulfonyl}ethyl]amino}ethyl]-2(3H)-benzothiazolone,1-(1-fluoro-4-hydroxyphenyl)-2-[4-(1-benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4(1-benzimidazolyl)-2-methyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminophenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-{4-[3-(4-methoxyphenyl)-1,2,4-triazol-3-yl]-2-methyl-2-butylamino}ethanol,5-hydroxy-8-(1-hydroxy-2-isopropylaminobutyl)-2H-1,4-benzoxazin-3-(4H)-one,1-(4-amino-3-chloro-5-trifluoromethylphenyl)-2-tert-butylamino)ethanol,1-(4-ethoxycarbonylamino-3-cyano-5-fluorophenyl)-2-(tert-butylamino)ethanol,and combinations thereof, each of which is optionally in the form of aracemate, enantiomer, diastereomer, or mixtures thereof, and alsooptionally in the form of a pharmacologically-compatible acid additionsalt.

Suitable corticosteroids and glucocorticoids for use in the presentinvention include, but are not limited to, prednisolone,methylprednisolone, dexamethasone, naflocort, deflazacort, halopredoneacetate, budesonide, beclomethasone dipropionate, hydrocortisone,triamcinolone acetonide, fluocinolone acetonide, fluocinonide,clocortolone pivalate, methylprednisolone aceponate, dexamethasonepalmitoate, tipredane, hydrocortisone aceponate, prednicarbate,alclometasone dipropionate, halometasone, methylprednisolonesuleptanate, mometasone furoate, rimexolone, prednisolone farnesylate,ciclesonide, deprodone propionate, fluticasone propionate, halobetasolpropionate, loteprednol etabonate, betamethasone butyrate propionate,flunisolide, prednisone, dexamethasone sodium phosphate, triamcinolone,betamethasone 17-valerate, betamethasone, betamethasone dipropionate,hydrocortisone acetate, hydrocortisone sodium succinate, prednisolonesodium phosphate, hydrocortisone probutate and combinations thereof.

Suitable LTD4 antagonists for use in the present invention include, butare not limited to, tomelukast, ibudilast, pobilukast, pranlukasthydrate, zafirlukast, ritolukast, verlukast, sulukast, cinalukast,iralukast sodium, montelukast sodium,4-[4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propylsulfonyl]phenyl]-4-oxobutyricacid,[[5-[[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propyl]thio]-1,3,4-thiadiazol-2-yl]thio]aceticacid,9-[(4-Acetyl-3-hydroxy-2-n-propylphenoxy)methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one,5-[3-[2-(7-Chloroquinolin-2-yl)vinyl]phenyl]-8-(N,N-dimethylcarbamoyl)-4,6-dithiaoctanoicacid sodium salt;3-[1-[3-[2-(7-Chloroquinolin-2-yl)vinyl]phenyl]-1-[3-(dimethylamino)-3-oxopropylsulfanyl]methylsulfanyl]propionicacid sodium salt,6-(2-Cyclohexylethyl)-[1,3,4]thiadiazolo[3,2-a]-1,2,3-triazolo[4,5-d]pyrimidin-9(1H-one,4-[6-Acetyl-3-[3-(4-acetyl-3-hydroxy-2-propylphenylthio)propoxy]-2-propylphenoxy]butyricacid,(R)-3-Methoxy-4-[1-methyl-5-[N-(2-methyl-4,4,4-trifluorobutyl)carbamoyl]indol-3-ylmethyl]-N-(2-methylphenylsulfonyl)benzamide,(R)-3-[2-Methoxy-4-[N-(2-methylphenylsulfonyl)carbamoyl]benzyl]-1-methyl-N-(4,4,4-trifluoro-2-methylbutyl)indole-5-carboxamide,(+)-4(S)-(4-Carboxyphenylthio)-7-[4-(4-phenoxybutoxy)phenyl]-5(Z)-heptenoicacid, compounds International Application No. PCT/EP03/12581, andcombinations thereof.

Suitable inhibitors of egfr-kinase for use in the present inventioninclude, but are not limited to, palifermin, cetuximab, gefitinib,repifermin, erlotinib hydrochloride, canertinib dihydrochloride,lapatinib,N-[4-(3-Chloro-4-fluorophenylamino)-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)-2(E)-butenamide,and combinations thereof.

Suitable p38 kinase inhibitors for use in the present invention include,but are not limited to, chlormethiazole edisylate, doramapimod,5-(2,6-Dichlorophenyl)-2-(2,4-difluorophenylsulfanyl)-6H-pyrimido[3,4-b]pyridazin-6-one,4-Acetamido-N-(tert-butyl)benzamide, SCIO-469 (described in Abst. ofClin. Pharmacol. Ther., 75(2), 2004, PII-7 and VX-702 (described inCirculation, 108 (17, Suppl. 4), Abst 882, 2003), and combinationsthereof.

Suitable NK1-receptor antagonists for use in the present inventioninclude, but are not limited to, nolpitantium besilate, dapitant,lanepitant, vofopitant hydrochloride, aprepitant, eziopitant,N-[3-(2-Pentylphenyl)propionyl]-threonyl-N-methyl-2,3-dehydrotyrosyl-leucyl-D-phenylalanyl-allo-threonyl-asparaginyl-serineC-1.7-O-3.1 lactone,1-Methylindol-3-ylcarbonyl-[4(R)-hydroxyl-L-prolyl-(3-(2-naphthyl)]-L-alanineN-benzyl-N-methylamide,(+)-(2S,3S)-3-[2-Methoxy-5-(trifluoromethoxy)benzylamino]-2-phenylpiperidine,(2R,4S)—N-[1-[3,5-Bis(trifluoromethyl)benzoyl]-2-(4-chlorobenzyl)piperidin-4-yl]quinoline-4-carboxamide,3-[2(R)-[1(R)-[3,5-Bis(trifluoromethyl)phenyl]ethoxy]3(S)-(4-fluorophenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-1H-1,2,4-triazole-1-phosphinicacid bis(N-methyl-D-glucamine) salt;[3-(2(R)-[1(R)-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3(S)-(4-fluorophenyl)-4-morpholinylmethyl)-2,5-dihydro-5-oxo-1H-1,2,4-triazol-1-yl]phosphonicacid 1-deoxy-1-(methylamino)-D-glucitol (1:2) salt,1′-[2-[2(R)-(3,4-Dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl]spiro[benzo[c]thiophen-1(3H)-4′-piperidine]2(S)-oxide hydrochloride and the compound CS-003 (described in Eur.Respir. J., 22(Suppl. 45): Abst P2664, 2003) and combinations thereof.

Suitable NSAIDs for use in the present invention include, but are notlimited to, Aceclofenac, acemetacin, acetylsalicylic acid, alclofenac,alminoprofen, amfenac, Ampiroxicam Antolmetinguacil, Anirolac,antrafenine, azapropazone, benorylate, Bermoprofen, bindarit, bromfenac,bucloxic acid, Bucolom, Bufexamac, Bumadizon, butibufen, Butixirat,Carbasalatcalcium, carprofen, choline magnesium trisalicylate,celecoxib, Cinmetacin, Cinnoxicam, clidanac Clobuzarit Deboxamet,dexibuprofen, Dexketoprofen, diclofenac, diflunisal, droxicam, EltenacEnfenaminsaure Etersalat, etodolac, etofenamate, etoricoxib Feclobuzonfelbinac, fenbufen, fenclofenac, fenoprofen, fentiazac, FepradinolFeprazon, Flobufen, floctafenine, flufenamic acid, flufenisal,Flunoxaprofen, flurbiprofen, Flurbiprofenaxetil, Furofenac, Furprofen,Glucametacin, ibufenac, ibuprofen, Indobufen, indomethacin,Indometacinfarnesil, indoprofen, Isoxepac, Isoxicam, ketoprofen,ketorolac, lobenzarit, Lonazolac, lornoxicam, Loxoprofen, lumiracoxib,meclofenamic, Meclofen, mefenamic acid, meloxicam, mesalazine, MiroProfen, Mofezolac, nabumetone, naproxen, niflumic acid, olsalazine,oxaprozin, Oxipinac, oxyphenbutazone, parecoxib, phenylbutazone,Pelubiprofen, Pimeprofen, Pirazolac, Priroxicam, pirprofen, Pranoprofen,Prifelon, Prinomod, Proglumetacin, Proquazon, Protizininsaure,rofecoxib, Romazarit, salicylamide, salicylic acid, Salmi Stein,Salnacedin, salsalate, sulindac, sudoxicam, suprofen, Talniflumate,tenidap, Tenosal, tenoxicam, tepoxalin, tiaprofenic acid, Taramid,Tilnoprofenarbamel, timegadine, Tinoridin, Tiopinac, tolfenamic acid,tolmetin, Ufenamat, valdecoxib, Ximoprofen, zaltoprofen, Zoliprofen andcombinations thereof.

The combinations described herein may be used in the treatment of anydisorder which is susceptible to amelioration by simultaneous,concomitant or sequential inhibition of phosphodiesterase 4 (PDE4).Thus, the present invention also includes methods of treatment of thesedisorders, as well as the use of the combinations of the invention inthe manufacture of a medicament for the treatment of these disorders.

Preferred examples of such disorders are those respiratory diseases,wherein the use of bronchodilating agents is expected to have abeneficial effect, for example asthma, acute or chronic bronchitis,emphysema, or Chronic Obstructive Pulmonary Disease (COPD).

Micronization of Roflumilast N-Oxide

In one preferred embodiment, the roflumilast N-oxide to be administeredby inhalation is micronized. Micronization can be performed bymechanical impact (e.g., by the use of mills, such as hammer mills, pinmills or bead milling) or by impact via fluid energy (e.g., by jetmilling or the use of spiral jet mills or fluidized bed jet mills).Additional details on micronization and the micronization process areprovided in a Spring 2005 Pharmaceutical Manufacturing and PackagingSourcerarticle by J. M Larran, Journal of Pharmaceutical Processing,“Advances in Powder Micronization Technology in Pharmaceutical Industryby Hokosawa Micron Powder System;” Wikipedia entry on micronization; G.Gianola, “Micronization Systems—Innovative Equipment Design andApplications,” presentation at Advances in Pharmaceutical Processing(Somerset, N.J., 2012) and R. Smith, “Micronization of ActivePharmaceutical Ingredients to Nanometer Scale,” presentation at Advancesin Pharmaceutical Processing (Somerset, N.J., 2012).

Provided below is an exemplary method used for micronization ofroflumilast N-Oxide using an Alpine Spiral Jet Mill (HOSOKAWA). Theprocess involved the following steps

-   -   15 gm of Roflumilast N-oxide was weighed.    -   Main knob for compressed air supply was opened.    -   Adjusted venture pressure at 6.0 bar and ring pressure at 0.5        bar.    -   Roflumilast N-oxide was added at a feed rate of ˜200 mg/min        manually through the hopper.    -   After completion of micronization, main air supply knob was        turned off and micronized roflumilast N-oxide was collected

Yield: ˜9.0 g (˜60% yield). Particle Size Distribution (PSD) of D₉₀:3.021 μm; D₅₀: 1.564 μm and D₁₀:0.777 μm

EXAMPLES

The present invention is now further illustrated by means of thefollowing non-limiting examples.

The experiments below show that roflumilast-N-oxide can be effectivelydelivered by inhalation at low doses to achieve an equal or betterbiological effect than that observed for roflumilast administeredorally.

Example 1: Lung Microsome Stability of Roflumilast and RoflumilastN-oxide

Metabolic stability studies were conducted using rat, dog and human lungmicrosomes. The protocol for the studies with rat, dog, and human lungmicrosomes (from Xenotech, USA) is provided below.

0.4 mg protein was preincubated with 2 mM NADPH (cofactor) in phosphatebuffer (pH˜7.4) for 15 minutes at 370° C. and then added with 1 μM testitem and incubated further for 60 minutes in triplicate. The reactionmixture was terminated with methanol containing an internal standard andcentrifuged further to analyze the test item remaining in thesupernatant by LC-MS/MS. The percent parent compound remaining wascalculated in comparison with similar samples terminated at 0 minutes.Table 1 shows the lung metabolic stability data.

TABLE 1 Percent Parent Compound Remaining Human Human Non AdditionalTest Item Rat Dog Smoker Smoker Observation Roflumilast 97.1 97.4 98.899.8 No N-Oxide Formation Roflumilast- 100.0 98.3 99.3 91.5 NoRoflumilast N-Oxide Formation

Example 2: Pharmacokinetic (PK) Studies

Pulmonary Kinetics by Intratracheal (IT) Route

Male and female Wistar rats were weighed and randomized for groups forvarious time points. Roflumilast N-oxide was prepared as a suspension ina suitable vehicle for intratracheal administration. For intratrachealdosing, animals were anesthetized with ketamine (50 mg/kg; i.p.) androflumilast N-oxide was administered via an Intratracheal Microsprayer®Aerosolizer for Rat with a 0.5 ml glass syringe (Model IA-1B-R-GL500)(Penn Century, US). A volume level of 0.5 ml/kg was administered intothe airway system of the rat at a dose of 1.0 mg/kg for the single doseand multiple dose study. The animals were kept under normal regular dietconditions and rat chew diet was provided ad libitum throughout thestudy. The blood and lung samples (all collections each of 150 μl fromeach animal) were collected according to the sampling schedule. Bloodsamples were collected from orbital sinus into the microfuge tubescontaining dipotassium EDTA as an anticoagulant. Blood samples werecentrifuged immediately with a speed of 1000 g for 10 minutes at 4° C.and separated plasma samples were frozen at below −80° C. and storeduntil analysis. The plasma and lung concentrations of roflumilastN-oxide in all samples were analyzed by LC-MS/MS (using X-Calibur 2.0.7software) as per the established method. The samples below the lowerlimit of quantification (LOQ) were mentioned as (BLQ) below level ofquantification in raw data and the results were tabulated accordingly.Pharmacokinetic parameters C_(max), AUC_(0-t), AUC_(0-∞), T_(max),t_(1/2), Kel, VZ, and CLZ were estimated for the above concentrationsusing WinNonlin (Phoenix 6.1 software) and the results were tabulated.Pharmacokinetic parameters in plasma were calculated from concentrationdata as ng/ml. C_(max) and AUC values were reported in terms of ng/mland ng·h/ml, respectively.

Pharmacokinetic parameters in the lungs were calculated fromconcentration data as ng/g. C_(max) and AUC values were reported interms of ng/g and ng·h/g, respectively. The lung to plasma concentrationratio was calculated by assuming equal to 1 the plasma density. Thetime-mean plasma concentration plots for roflumilast N-oxide were doneusing Graph pad Prism 5.02 software. Table 2 shows the lung and plasmakinetics for the single and multiple dose studies.

TABLE 2 Single Dose N = 3 for each time point at dose of 1 mg/kg ofRoflumilast N-Oxide (IT) Lung Plasma Parameter Units N-Oxide RoflumilastUnits N-Oxide Roflumilast C_(max) ng/g 26350 No ng/ml 530 No AUC_(0-t)ng · hr/g 47880 Formation ng · hr/ml 4160 Formation AUC_(0-inf) ng ·hr/g 47960 ng · hr/m1 4350 T_(max) hr 0.25 hr 0.25 t_(1/2) hr 2.54 hr5.43 Multiple Dose N = 3 for each time point at dose of 1 mg/kg ofRoflumilast N-Oxide (IT) for 7 days Lung Plasma Day-1 Day-7 Day-1 Day-7N- N- N- N- Parameter Units Oxide Roflumilast Oxide Roflumilast UnitsOxide Roflumilast Oxide Roflumilast C_(min) μg/g — No 0.00 No μg/mL — No0.00 No C_(avg) μg/g — Formation 0.77 Formation μg/mL — Formation 0.12Formation C_(max) μg/g 26.35 23.15 μg/mL 0.53 0.56 AUC_(0-t) μg · hr/g47.88 17.96 μg · hr/ 4.16 2.25 mL AUC_(tau) μg · hr/g — 18.51 μg · hr/ —2.81 mL AUC_(0-inf) μg · hr/g 47.96 18.04 μg · hr/ 4.35 2.39 mL T_(max)hr  0.25 0.25 hr 0.25 1.00 t_(1/2) hr  2.54 0.81 hr 5.43 1.53

Example 3: Inhalation Studies

For inhalation studies, roflumilast N-Oxide is administered as asuspension. Roflumilast N-oxide (100 mg) is placed in a mortar andtriturated following addition of a suitable solvent in a gravimetricdilution to afford a fine suspension.

For inhalation suspension dosing, animals are placed in a plexiglasschamber and exposed to roflumilast N-oxide (at a suitable concentration)for a period of 30 minutes in an aerosol form using a Piston Nebulizer(Infi-Neb, USA) at an appropriate flow rate (L/min).

For dry powder inhalation dosing, animals are anesthetised with ketamine(50 mg/kg; i.p.), then the animals are administered a mixture ofmicronized roflumilast N-oxide and a suitable carrier in a powder formvia intratracheal insufflation (using a Penn Century insufflationspowder delivery device (DP-4, US)).

Example 3A: In Vitro Biological Studies

Lipopolysaccharide (LPS) Induced TNFα in MH-S (Mouse AlveolarMacrophage) Cells:

MH-S represents a mouse alveolar macrophage cell line that secretescopious amounts of TNFα upon induction by LPS. Cells are plated at150,000 cells per well. Different concentrations of roflumilast N-oxideare added 15 minutes prior to the addition of LPS. LPS (1 μg/ml) isadded and then incubated for 4 hours. The supernatant is collected atthe end of incubation period and TNFα estimated using an ELISA kit.Percent inhibition and IC₅₀ values are determined.

LPS Induced TNFα in THP-1 (Human Monocyte) Cells:

THP-1 represents a monocytic cell line that has elevated endogenous pAKTlevels and secretes copious amounts of TNFα upon induction by LPS. Cellsare plated at 150,000 cells per well. Different concentrations ofroflumilast N-oxide are added. LPS (1 μg/ml) is added and then incubatedfor 4 hours. The supernatant is collected at the end of incubationperiod and TNFα estimated using an ELISA kit. Percent inhibition andIC₅₀ values are determined.

Con A+PMA induced IFNγ in Human Whole Blood:

Freshly collected human whole blood (HWB) is diluted with media andincubated with a desired concentration of inhibitor for 15 minutes.Cytokine release is induced with the addition of Concanavalin A (25μg/ml)+Phorbol Myristate Acetate (50 ng/ml). The supernatant iscollected after 20 hours and IFNγ estimated using an ELISA kit. Percentinhibition and IC₅₀ values are determined.

Con A+PMA Induced IFNγ in Peripheral Blood Mononuclear Cells (PBMC):

PBMC from whole blood are isolated by density gradient using Histopaqueand incubated with a desired concentration of inhibitor for 15 minutes.Cytokine release is induced with the addition of Concanavalin A (25μg/ml)+Phorbol Myristate Acetate (50 ng/ml). The supernatant iscollected after 20 hours and IFNγ estimated using an ELISA kit. Percentinhibition and IC₅₀ values are determined.

Example 3B: In Vivo Biological Studies

A. Lipopolysaccharide (LPS) Induced Pulmonary Neutrophilia in a FemaleSprague-Dawley Rat Model

An exaggerated recruitment and subsequent activation of neutrophila islikely to be important for the development and course of severalinflammatory diseases in the airways and lungs, such as severe asthma,chronic obstructive pulmonary disease, cystic fibrosis, and acuterespiratory distress syndrome. The mechanisms by which neutrophilacontribute to these diseases may involve the release of proteolyticenzymes, such as neutrophil elastase, and free oxygen radicals. Whenreleased, these compounds can cause bronchoconstriction, bronchialhyperreactivity, hyper-secretion, epithelial damage, and tissueremodelling in the airways.

Roflumilast was prepared as a suspension in a suitable vehicle for oraladministration. For intratracheal administration, roflumilast orroflumilast N-oxide was prepared as a dry powder mixture andadministered by using a dry powder Insufflator (Penn-Century, USA)Animals were anaesthetized with ketamine and LPS solution wasadministered intratracheally one hour after roflumilast (at a dose of0.3, 1, 3, and 10 mg/kg orally), or 30 minutes after roflumilast (at adose of 10, 30 and 100 μg/kg i.t) or roflumilast N-oxide administration(at a dose of 10, 30 and 100 μg/kg, i.t). 6 hours after LPSinstillation, animals were exsanguinated under anaesthesia, and thentrachea were cannulated and the lungs were lavaged with 5-ml aliquots ofheparinised PBS (1 unit/ml) four times through tracheal cannula (totalvolume 20 ml). BAL (bronchoalveolar lavage) fluid was stored at 2-8° C.until assayed for total cell and differential leukocyte count.Bronchioalveolar fluid was centrifuged (500×g for 10 minutes) and theresulting cell pellet was resuspended in 0.5 ml of heparinized saline.The total number of white blood cells were determined in BAL fluid usinga blood cell counter and were adjusted to 1×10⁶ cell/ml. Differentialcell count was calculated manually. One hundred microliters of the cellsuspension was centrifuged using cytospin 3 to prepare a cell smear. Thecell smear was stained with a blood staining solution fordifferentiation and slides were microscopically observed to identifyeosinophil according to their morphological characteristics. The numberof each cell type among 300 white blood cells in the cell smear wasdetermined and expressed as a percentage. The total number ofneutrophils in each BAL fluid was calculated.

Roflumilast and roflumilast N-oxide were administered intratracheally(i.t) at doses of 100, 30, and 10 μg/kg as 1:200; 1:500; and 1:1000drug:lactose blends, respectively.

i. Effective Dose of Roflumilast

Roflumilast upon oral administration demonstrated a dose dependentinhibition in neutrophil infiltration compared to the control group at0.3, 1, 3 and 10 mg/kg. The results are shown in FIG. 1. Percentinhibitions were −7.89%, 43.46%, 68.02%, and 92.21%, respectively, andthe 50% inhibition (ED₅₀) dose was 1.8 mg/kg.

Roflumilast upon intratracheal administration demonstrated a dosedependent inhibition in neutrophil infiltration compared to the controlgroup at 10, 30 and 100 μg/kg. The results are shown in FIG. 2. Percentinhibitions were 31.76%, 60.47%, and 64.40%, respectively, and the 50%inhibition (ED₅₀) dose was 26 μg/kg.

ii. Effective Dose of Roflumilast N-Oxide

Roflumilast N-oxide upon intratracheal administration demonstrated adose dependent inhibition in neutrophil infiltration compared to controlgroup at 10, 30 and 100 μg/kg. The results are shown in FIG. 3. Percentinhibitions were 37.52%, 49.66%, and 69.48%, respectively, and the 50%inhibition (ED₅₀) dose was 27 μg/kg.

B. Acute Cigarette Smoke Induced Cell Infiltration in Female Balb/c Mice

Animals were acclimatized for seven days prior to the start of theexperiment. The animals were randomly distributed to various groupsbased on their body weight. For oral administration of roflumilast andfor intranasal administration of roflumilast N-oxide, roflumilast orroflumilast N-oxide were prepared as a suspension in a suitable vehicle.On day 1, mice were administered roflumilast N-oxide or roflumilast andafter 1 hour, the animals were placed in a whole body exposure box. Onday 1 and day 2, the mice were exposed to the mainstream smoke of 6cigarettes, and 8 cigarettes on day 3 and day 4. Exposure to the smokeof each cigarette lasted for 10 minutes (cigarettes were completelyburned in the first two minutes and followed by an air flow with animalventilator, then 20 minutes exposure to fresh room air). After everysecond cigarette an additional break of 20 minutes with exposure tofresh room air was conducted. Control animals were exposed to room airchamber. From day 1 to day 4, the animals were administered roflumilastN-oxide or roflumilast. On day 5, 24 hours after the last cigarettesmoke (CS) exposure, the animals were exsanguinated under anaesthesia,the trachea were cannulated and the lungs were lavaged with 0.5-mlaliquots of heparinised PBS (1 unit/ml) four times through trachealcannula (total volume 2 ml). Bronchioalveolar (BAL) collected was storedat 2-8° C. until assayed for total cell and differential leukocytecount. BAL fluid was centrifuged (500×g for 10 minutes) and theresulting cell pellet was resuspended in 0.5 ml of heparinised saline.The total number of white blood cells was determined in BAL fluid andadjusted to 1×10⁶ cell/ml. Differential cell count was calculatedmanually by diff-quick staining. Forty microliters of the cellsuspension was centrifuged using cytospin 3 to prepare a cell smear. Thecell smear was stained with a blood staining solution fordifferentiation and microscopically observed to identify cell typeaccording to their morphological characteristics. The number of eachcell type among 300 white blood cells in the cell smear were determinedand expressed as a percentage, and the number of neutrophils andmacrophages in each BAL fluid were calculated.

Roflumilast and roflumilast N-oxide as a suspension in polysorbate 80(1% v/v) and methyl cellulose (MC) (0.5% w/v) were used for oral andintranasal administration respectively.

i. Effective Dose of Roflumilast

Roflumilast upon oral administration demonstrated a dose dependentinhibition in macrophage infiltration compared to control group at 1, 3and 10 mg/kg. Percent inhibitions were 22.2%, 51.00%, and 69.11%,respectively, and the 50% inhibition (ED₅₀) dose was 3.5 mg/kg. Dosedependent inhibition in neutrophil infiltration was observed and thepercent inhibitions were 70.85%, 73.69%, and 83.01% respectively, andthe 50% inhibition (ED₅₀) dose was 1.75 mg/kg. The results formacrophages and neutrophils are shown in FIGS. 4A and 4B, respectively.

ii. Effective Dose of Roflumilast N-Oxide

Roflumilast N-oxide upon intranasal administration demonstrated a dosedependent inhibition in macrophage infiltration compared to controlgroup at 0.003, 0.03, 0.3 and 3 mg/kg. Percent inhibitions were 50.68%,55.10%, 62.79% and 63.35%, respectively, and the 50% inhibition (ED₅₀)dose was 0.049 mg/kg. Dose dependent inhibition in neutrophilinfiltration was observed and the percent inhibitions were 26.60%,59.28%, 66.49% and 72.08% respectively. The 50% inhibition (ED₅₀) dosewas 0.038 mg/kg. The results for macrophages and neutrophils are shownin FIGS. 5A and 5B, respectively.

C. Ovalbumin Induced Pulmonary Eosinophilia in Male Guinea Pigs

Airway inflammation and hyper-responsiveness (AHR) are hallmarks anddistinguishing features of bronchial asthma. Provocation ofpre-sensitized mice with the same allergen induces airway inflammationwith preferential eosinophilic infiltration and, as a consequence, AHR.Pulmonary eosinophilia and airway remodelling in conjunction withaltered neural control of airway tone and airway epithelial desquamationmay contribute to AHR in asthma.

After the quarantine period, 0.3 mL blood samples are collected fromorbital vein by retro-orbital plexus method from each individual animaland analysed on a cell analyser (ADVIA 2120, Siemens). Based on theirtotal cell count, guinea pigs are randomized and divided into variousgroups. Ear pinna is marked with an indelible marking pen foridentification. On day 0, weights are recorded and the animals are thensensitized with 50 μg of Ovalbumin and 10 mg of alum solution (1 mL)intraperitoneally. On day 7 and day 14, the above sensitization protocolis repeated. Roflumilast N-oxide is prepared as a suspension in asuitable vehicle or as a dry powder mixture. Roflumilast N-oxide orvehicle is administered by aerosol suspension or dry powderinsufflation. On day 18, the animals are treated with roflumilastN-oxide. On days 19 and 20, the animals are treated with roflumilastN-oxide and exposed to 0.5% w/v ovalbumin for 10 minutes using anultrasonic nebulizer with a flow rate of 0.2 ml per minute. On day 21,fasted animals are treated with roflumilast N-oxide and 15 minutes afterdosing, animals are nebulized with 1% w/v ovalbumin solution for 10 minControl group animals are treated with 0.5% w/v methyl cellulose(vehicle). Sham control groups are sensitized with 10 mg of alum on days0, 7 and 14 and exposed to saline solution with the same nebulizationrate on days 19, 20 and 21. Twenty four hours after OVA challenge, bloodsamples and BAL fluid is collected. Samples are analysed for total cellcount using a blood analyser (ADVIA 2120, Siemens) and differentialleukocyte count is performed manually.

Example 4: Safety and Toxicity

In order to determine the safety of roflumilast N-oxide by inhalationcompared to roflumilast by oral administration, a 7- and 14-day repeatdose safety study was conducted according to the protocol given in Table5 below.

TABLE 5 Species: Wistar Rats Experimental design 5M + 5F - Main studyand 5M + 5F in placebo groups Compound Roflumilast N-Oxide RoflumilastDose 1000 ug/kg/day 10 mg/Kg/day Route Intratracheal Oral ED50 in 27ug/kg 1.8 mg/kg Lipopolysaccharide induced pulmonary neutrophilia infemale Sprague-Dawley rat model Criteria for Dose ~37 fold to ED50 of~5.5 fold to ED50 of selection: Roflumilast N-oxide Roflumilast Durationof the 7 & 14 days for 7 days for Roflumilast treatment: RoflumilastN-oxide

The results are presented in Table 6.

TABLE 6 STUDY 7-DAY REPEAT DOSE 14-DAY REPEAT DOSE Drug & RouteRoflumilast N-Oxide Roflumilast N-Oxide PARAMETERS Roflumilast by Oral*by Intratracheal by Intratracheal CLINICAL SIGNS Abnormal clinical Noabnormal clinical No abnormal clinical signs were observed in signs wereobserved signs were observed in treatment group in treatment grouptreatment group animals compared to animals compared to animals comparedto placebo group animals placebo group animals placebo group animals(abnormal gait) MORTALITY 1M animal died on day 7 No Mortality NoMortality BODY WEIGHT Yes, significant No significant No significantPROFILE reduction in body reduction in body reduction in body weight ofaround 25% weight observed at all weight observed at all observed in alltreatment groups treatment groups treatment groups (n = 10) HEMATOLOGYYes, elevated WBC No treatment related No treatment related with acorresponding changes observed changes observed lymphocytopenia acrossthe groups. across the groups. CLINICAL Yes, reduced serum No abnormalclinical No abnormal clinical CHEMISTRY albumin and ALP, signs wereobserved signs were observed in increases in total in treatment grouptreatment group bilirubin animals compared to animals compared toplacebo group animals placebo group animals ORGAN WEIGHTS Yes, reducedthymus No differences in the No differences in the and spleen weightwith absolute and relative absolute and relative an increase in weightorgan weight to body organ weight to body of adrenals weight betweenweight between control and treatment control and treatment group animalsgroup animals GROSS PATHOLOGY Yes, treatment related No treatmentrelated No treatment related gross pathological gross pathological grosspathological changes including changes observed changes observed stomachenlargement across the treatment across the treatment groups groupsHISTOPATHOLOGY Infiltration of No significant No significant neutrophilsand histopathological histopathological mononuclear cells into changesobserved in changes observed in lung alveolus observed lung, stomach,spleen, lung, stomach, spleen, indicating significant thymus andadrenal. thymus and adrenal lung damage. Other changes included atrophyof thymus and spleen, hypertrophy/ hyperplasia of adrenals and gastriculcerations.

Intratracheal administration of roflumilast N-oxide was found to be safewhen compared to oral administration of roflumilast, which showedsignificant toxicity.

Intratracheal administration of roflumilast N-oxide (1000 μg/kg) wasfound to be safe and provide a significant therapeutic window of morethan 37-fold based upon its ED₅₀ of 27 μg/kg. In contrast, oraladministration of roflumilast (10 mg/kg) had a lower therapeutic indexgiven its ED₅₀ of 1.8 mg/kg (i.e., it is toxic even at a 5.5 fold).

The preceding data on metabolic stability, pharmacokinetics, in vivoefficacy and safety studies related to roflumilast N-oxide demonstratesthat administration of roflumilast N-oxide by inhalation has the desiredtherapeutic effect with significant improvements in safety when comparedto oral administration of roflumilast.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as described above. It is intended that theappended claims define the scope of the invention and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

All publications, patents and patent applications cited in thisapplication are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference.

1. A method of treating an autoimmune, respiratory or inflammatorydisease or condition in a subject in need thereof comprising pulmonaryadministration of an effective amount of roflumilast N-oxide or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the roflumilast N-oxide or pharmaceutically acceptable saltthereof is administered by inhalation.
 3. The method of claim 1, whereinthe roflumilast N-oxide or pharmaceutically acceptable salt thereof isadministered by inhalation as a dry powder, solution or suspension. 4.The method of claim 3, wherein the roflumilast N-oxide orpharmaceutically acceptable salt thereof is administered as a drypowder.
 5. The method of claim 3, wherein roflumilast N-oxide orpharmaceutically acceptable salt thereof is administered as a solutionor suspension.
 6. The method of claim 1, wherein the roflumilast N-oxideor pharmaceutically acceptable salt thereof is administered at a singledose of about 5 μg to about 2000 μg.
 7. The method of claim 6, whereinthe roflumilast N-oxide or pharmaceutically acceptable salt thereof isadministered at a single dose of about 20 μg to about 1200 μg.
 8. Themethod of claim 6, wherein the roflumilast N-oxide or pharmaceuticallyacceptable salt thereof is administered at a single dose of 50 μg to1000 μg.
 9. The method of claim 6, wherein the roflumilast N-oxide orpharmaceutically acceptable salt thereof is administered at a singledose of 100 μg to 800 μg.
 10. The method of claim 6, wherein theroflumilast N-oxide or pharmaceutically acceptable salt thereof isadministered at a single dose of 100 μg, 200 μg, 400 μg, or 600 μg. 11.The method of claim 1, wherein the disease or condition is selected fromasthma, COPD, chronic obstructive bronchiolitis, acute bronchitis,chronic bronchitis, emphysema, allergic rhinitis and non-allergicrhinitis.
 12. The method of claim 1, wherein the disease or condition isasthma.
 13. The method of claim 1, wherein the disease or condition isCOPD.
 14. The method of claim 1, wherein the roflumilast N-oxide orpharmaceutically acceptable salt thereof is administered in combinationwith a long-acting β2 agonist, an M3 antagonist, a corticosteroid, orany combination thereof.
 15. The method of claim 12, wherein thelong-acting β2 agonist is selected from carmoterol, GSK-642444,indacaterol, milveterol, arformoterol, formoterol, salbutamol,formoterol, levalbuterol, terbutaline, AZD-3199, BI-1744-CL, LAS-100977,bambuterol, isoproterenol, procaterol, clenbuterol, reproterol,fenoterol, ASF-1020, and any combination thereof.
 16. The method ofclaim 14, wherein the M3 antagonist is selected from aclidinium,tiotropium, ipratropium and oxitropium, and any combination thereof. 17.The method of claim 14, wherein the corticosteroid is selected from thegroup consisting of dexamethasone, fluticasone, fluticasone furoate,prednisolone, betamethasone, budesonide, mometasone, mometasone furoate,triamcinolone acetonide, ciclesonide, TPI-1020, beclomethasone,beclomethasone dipropionate, prednisone, deflazacort, hydrocortisone,QAE-397, flunisolide, and any combination thereof.
 18. The method ofclaim 1, wherein the roflumilast N-oxide or pharmaceutically acceptablesalt thereof is anhydrous, a solvate, or a hydrate.
 19. RoflumilastN-oxide having a d₅₀ or d₉₀ of less than about 10 microns.
 20. Theroflumilast N-oxide of claim 19, wherein the d₅₀ or d₉₀ is (i) equal toor less than about 10 microns, (ii) less than about 6 microns, or (iii)between about 1 and about 6 microns.
 21. The roflumilast N-oxide ofclaim 19, wherein the roflumilast N-oxide is suitable for inhalation.22. The roflumilast N-oxide of claim 21, having a d₅₀ or d₉₀ of (i) lessthan about 6 microns or (ii) between about 1 and about 6 microns.
 23. Apharmaceutical composition comprising roflumilast N-oxide and,optionally, one or more pharmaceutically acceptable carriers and/orexcipients, wherein the composition is administrated by inhalation. 24.The pharmaceutical composition of claim 23, in the form of a metereddose inhaler (MDI) or a dry powder inhaler (DPI).
 25. The pharmaceuticalcomposition of claim 23, wherein the pharmaceutical composition is inthe form of an inhalable dry powder comprising micronized particles ofroflumilast N-oxide or a pharmaceutically acceptable salt thereof as anactive ingredient and particles of a physiologically acceptablepharmacologically-inert solid carrier.
 26. The pharmaceuticalcomposition of claim 23, wherein the pharmaceutical composition is inthe form of an aerosol comprising roflumilast N-oxide or apharmaceutically acceptable salt thereof as an active ingredient, apropellant and, optionally, one or more co-solvents, pharmaceuticallyacceptable carriers and/or excipients.
 27. The pharmaceuticalcomposition of claim 26, wherein the roflumilast N-oxide is in the formof a suspension of particles of micronized roflumilast N-oxide in thepropellant.
 28. The pharmaceutical composition of claim 23, wherein thed₅₀ or d₉₀ of the roflumilast N-oxide is (i) about 10 microns or less,(ii) less than about 6 microns or (iii) between about 1 and about 6microns.
 29. The pharmaceutical composition of claim 23, furthercomprising a long-acting β2 agonist, an M3 agonist, a corticosteroid, orany combination thereof.
 30. The pharmaceutical composition of claim 29,wherein the long-acting β2 agonist is selected from carmoterol,GSK-642444, indacaterol, milveterol, arformoterol, formoterol,salbutamol, formoterol, levalbuterol, terbutaline, AZD-3199, BI-1744-CL,LAS-100977, bambuterol, isoproterenol, procaterol, clenbuterol,reproterol, fenoterol, ASF-1020, and any combination thereof.
 31. Thepharmaceutical composition of claim 29, wherein the M3 antagonist isselected from aclidinium, tiotropium, ipratropium and oxitropium, andany combination thereof.
 32. The pharmaceutical composition of claim 29,wherein the corticosteroid is selected from the group consisting ofdexamethasone, fluticasone, fluticasone furoate, prednisolone,betamethasone, budesonide, mometasone, mometasone furoate, triamcinoloneacetonide, ciclesonide, TPI-1020, beclomethasone, beclomethasonedipropionate, prednisone, deflazacort, hydrocortisone, QAE-397,flunisolide, and any combination thereof.
 33. The pharmaceuticalcomposition of claim 23, wherein the roflumilast N-oxide orpharmaceutically acceptable salt thereof is anhydrous, a solvate, or ahydrate.